Enteropathogenic E. coli (EPEC), an important food-borne pathogen, causes infantile diarrhea resulting in significant morbidity and mortality. However, the mechanisms of EPEC-induced diarrhea remain unclear. Diarrhea results from either increased secretion, decreased absorption, or both. Earlier studies have failed to conclusively show an increase in host secretory response by EPEC infection. Our preliminary data utilizing Caco-2 cells, showed that EPEC infection significantly inhibited the activities of both the predominant Na+ absorbing isoform NHE3, the CI-OH exchanger and butyrate uptake. An analysis of the EPEC effects on NHEs showed that NHE1 and NHE2 activities were rapidly increased while the activity of NHE3 was significantly decreased. We hypothesize that one of the potential mechanisms of EPEC-induced diarrhea involves a decrease in intestinal absorptive processes. Our proposed studies will explore the effects of EPEC infection on intestinal NHE activities both in in vitro and an in vivo model and elucidate the mechanisms(s) underlying the differential regulation of NHEs by EPEC. Our Specific Aims are designed to: 1. To dissect the effects of EPEC on NHE isoform activities and Na+ flux in model human small intestinal (Caco-2.bbe) and non-transformed colonic (NCM460) epithelia; 2. Define mechanisms of EPEC-induced modulation of NHE isoform activities by elucidating signaling pathways involved, the role of regulatory factors (NHERF1 and NHERF2), the role of the cytoskeletal protein ezrin, and NHE membrane trafficking; 3. Examine the effects of EPEC on Na+ transport in ileum and colon in murine model of EPEC infection and in NHE2 & 3 knockout mice by determining the expression and activities of NHEs, transepithelial Na+ fluxes, gut luminal fluid accumulation, and the role of EPEC virulence genes. The results of these studies will enhance our understanding of the mechanisms of regulation of human intestinal NHEs and their modulation by pathogenic organisms. Our findings may aid in the future development of improved therapeutic modalities for infectious diarrhea.